The need to determine multiple nucleic acid sequences in biological samples at the same time in an efficient, reliable and reproducible manner has become increasingly important to numerous disciplines. Most multi-target analyses, such as assays that detect multiple nucleic acid sequences, involve several steps and/or require multiple reactions, have poor sensitivity, a limited dynamic range (typically on the order of 2 to 100-fold differences) and some require sophisticated instrumentation.
At present, there are a large number of application for assays useful to determine the presence of specific sequences, distinguishing between alleles in homozygotes and heterozygotes, determining the presence of mutations, evaluating cellular expressions patterns, etc. In many of these cases one will wish to determine in a single reaction, a number of different characteristics of the same sample. In many assays, there will be an interest in determining the presence of specific sequences, whether genomic, synthetic, or cDNA. Any method should be accurate, have reasonable associated costs and provide for a multiplexed assay, which allows for differentiation and quantitation of multiple genes, and/or SNP determination, and/or gene expression.
At present, when analyzing large and/or undefined genes, multiple individual PCR reactions are often required to identify critical base changes or deletions. The results obtained with multiplex PCR are frequently complicated by artifacts of the amplification procedure including both “false-negatives” and “false-positives”. The use of the Amplification Refractory Mutation system (ARMS), also known as Allele Specific PCR (ASPCR) and PCR Amplification of Specific Alleles (PASA) has been described for detection of known single-base substitutions or mocrodeletions/insertions. See, e.g., Sommer et al. Mayo Clinic Proc 64, 1361, 1989 ( PASA); Netwon et al. Nucl Acids Res 17, 2503, 1989 (ARMS); and Wu et al. PNSA 86, 2757, 1989 (ASPCR). The assay typically requires two separate reactions to determine both alleles for a given gene locus.
Thus an assay that can be used to differentiate and quantiate multiple genes, and/or be used to analyze gene expression at the RNA level is desirable. Preferably such an assay has most or all of the following characteristics: (1) a simple format, such as a single assay mixture where most or all of the reagents are added at the same time; (2) high sensitivity; (3) a large dynamic range (103 to 104-fold differences in target levels); (4) multiplexing capability; (5) reproducibility/reliability; and (6) a reasonable cost per analysis.